Abstract [en]

As multidrug resistance in Gram-negative bacilli increases, the old antibiotic colistin has rapidly gained attention as one of few last line treatment options in the form of colistin methanesulfonate (CMS), which is hydrolyzed to colistin both in vitro and in vivo. There is a dearth of knowledge on fundamental aspects of colistin, including pharmacokinetics and optimal dosing regimens. The aim of this thesis was to improve the basis for optimal colistin therapy.

To be able to study colistin, an LC-MS/MS assay method was developed which is sensitive, specific and useful in both in vivo and in vitro studies. Using this method we detected a significant loss of colistin during standard laboratory procedures. This loss was characterized and quantified, the hypothesis being that the loss is mainly caused by adsorption to labware.

The pharmacokinetics of colistin was studied in two populations of critically ill patients, one with normal renal function and one with renal replacement therapy. Plasma concentrations were assayed with the method above, and population modeling was employed to describe the data. The results include a previously unseen, long elimination half-life of colistin. The data from the population on renal replacement therapy was described without modeling, and showed that both CMS and colistin are cleared by hemodiafiltration.

Combination therapy is an approach that is often used when treating patients infected with multidrug-resistant pathogens. The thesis discusses how the joint effect of antibiotics can be measured using colistin and meropenem as a model, and proposes a method for testing antibiotic combinations. Furthermore, a PKPD model was adapted to describe the pharmacodynamics of the combination.

In conclusion, a specific and sensitive method for analysis of colistin was developed and the adsorption of colistin to materials was described. The assay method has been well accepted internationally. The pharmacokinetics of colistin and CMS was described in two important patient populations, partly with surprising results that have influenced dosages of colistin worldwide. The pharmacodynamics of combination therapy was investigated and quantified, and the methods applied could be further developed into clinically useful tools for selection of antibiotic combinations.

Abstract [en]

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm x 250 mm, 5 mu m particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor-product ion pairs were m/z 1167.7 -> 1079.6 for colistin A and m/z 1153.7 -> 1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 mu L plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV <6.2% and accuracy <+/- 12.6%. For culture medium (50 mu L+ 50 mu L plasma), LLOQ was 24.2 and 13.2 ng/mL for colistin A and B, respectively, with CV <11.4% and accuracy <+/- 8.1%. The quick sample work-up method allows for determination of colistin A and B in clinical samples without causing hydrolysis of the prodrug colistin methanesulfonate (CMS).

Abstract [en]

Colistin adheres to a range of materials, including plastics in labware. The loss caused by adhesion influences an array of methods detrimentally, including MIC assays and in vitro time-kill experiments. The aim of this study was to characterize the extent and time course of colistin loss in different types of laboratory materials during a simulated time-kill experiment without bacteria or plasma proteins present. Three types of commonly used large test tubes, i.e., soda-lime glass, polypropylene, and polystyrene, were studied, as well as two different polystyrene microplates and low-protein-binding microtubes. The tested concentration range was 0.125 to 8 mg/liter colistin base. Exponential one-phase and two-phase functions were fitted to the data, and the adsorption of colistin to the materials was modeled with the Langmuir adsorption model. In the large test tubes, the measured start concentrations ranged between 44 and 102% of the expected values, and after 24 h, the concentrations ranged between 8 and 90%. The half-lives of colistin loss were 0.9 to 12 h. The maximum binding capacities of the three materials ranged between 0.4 and 1.1 μg/cm2, and the equilibrium constants ranged between 0.10 and 0.54 ml/μg. The low-protein-binding microtubes showed start concentrations between 63 and 99% and concentrations at 24 h of between 59 and 90%. In one of the microplates, the start concentrations were below the lower limit of quantification at worst. In conclusion, to minimize the effect of colistin loss due to adsorption, our study indicates that low-protein-binding polypropylene should be used when possible for measuring colistin concentrations in experimental settings, and the results discourage the use of polystyrene. Furthermore, when diluting colistin in protein-free media, the number of dilution steps should be minimized.

Open this publication in new window or tab >>Population pharmacokinetic analysis of colistin methanesulfonate and colistin after intravenous administration in critically ill patients with infections caused by gram-negative bacteria

Giamarellou, H

Abstract [en]

Colistin is used to treat infections caused by multidrug-resistant gram-negative bacteria (MDR-GNB). It is administered intravenously in the form of colistin methanesulfonate (CMS), which is hydrolyzed in vivo to the active drug. However, pharmacokinetic data are limited. The aim of the present study was to characterize the pharmacokinetics of CMS and colistin in a population of critically ill patients. Patients receiving colistin for the treatment of infections caused by MDR-GNB were enrolled in the study; however, patients receiving a renal replacement therapy were excluded. CMS was administered at a dose of 3 million units (240 mg) every 8 h. Venous blood was collected immediately before and at multiple occasions after the first and the fourth infusions. Plasma CMS and colistin concentrations were determined by a novel liquid chromatography-tandem mass spectrometry method after a rapid precipitation step that avoids the significant degradation of CMS and colistin. Population pharmacokinetic analysis was performed with the NONMEM program. Eighteen patients (6 females; mean age, 63.6 years; mean creatinine clearance, 82.3 ml/min) were included in the study. For CMS, a two-compartment model best described the pharmacokinetics, and the half-lives of the two phases were estimated to be 0.046 h and 2.3 h, respectively. The clearance of CMS was 13.7 liters/h. For colistin, a one-compartment model was sufficient to describe the data, and the estimated half-life was 14.4 h. The predicted maximum concentrations of drug in plasma were 0.60 mg/liter and 2.3 mg/liter for the first dose and at steady state, respectively. Colistin displayed a half-life that was significantly long in relation to the dosing interval. The implications of these findings are that the plasma colistin concentrations are insufficient before steady state and raise the question of whether the administration of a loading dose would benefit critically ill patients.

Giamarellou, Helen

Abstract [en]

This report describes the pharmacokinetics of colistin methanesulfonate (CMS) and colistin in five intensive care unit patients receiving continuous venovenous hemodiafiltration. For CMS, the mean maximum concentration of drug in plasma (C(max)) after the fourth dose was 6.92 mg/liter and total clearance (CL) 8.23 liters/h. For colistin, the mean concentration was 0.92 mg/liter and CL/metabolized fraction (f(m)) 18.91 liters/h. Colistin concentrations were below the current MIC breakpoints, and the area under the concentration-time curve for the free, unbound fraction of the drug over 24 h in the steady state divided by the MIC (fAUC/MIC) was lower than recommended, suggesting that a dosage regimen of 160 mg CMS every 8 h (q8h) is inadequate.

Cars, Otto

Abstract [en]

As multidrug resistance in Gram-negative bacilli increases, colistin has rapidly gained much attention as one of few last line treatment options. An important part of therapeutic regimens with colistin has been the use of combinations, although there are no reliable methods available for testing combinations, and that the interpretation of joint effects is lacking. In addition, results of combination tests have been varying, with some results indicating no benefit of combinations while others indicate the opposite. The aims of this study were to describe the PD of colistin and meropenem in combination using clinically relevant concentrations of the drugs, to investigate some key endpoints for measuring effect, and to propose a possible path toward testing clinical strains with regard to joint effects.

Eight isolates of P. aeruginosa and A. baumannii, with and without meropenem resistance, were exposed to meropenem and colistin separately and in combination over one dosage interval in an in vitro kinetic model. The PD endpoints were initial kill rate, maximum kill, net kill and area under the bactericidal curve (AUBC). Checkerboard assays were performed for comparison.

Compared to the single drug exposures, the combination increased the effect with all endpoints in three of the strains, including one resistant to meropenem. The AUBC was significantly (paired t-test, p<0.05) lowered in three of the strains.

The chosen endpoints reflect different features of drug effect, and may be important to discern in some patient populations. Kill rate, maximum kill and net kill are point-based endpoints and focusing on only one of them (e.g. net kill by tradition) lead to wasting of potentially important information. AUBC reflects a compressed measure of the whole dosage interval that probably is relevant to most patients and can be used in evaluating joint effect easily and robustly by a simple t-test. The combination of meropenem and colistin is a useful option for treatment of multidrug resistant infections.